UCSF

The post-translational modification of serines and threonines of intracellular proteins by N-acetylglucosamine (O-GlcNAc) is becoming increasing implicated in processes which control embryonic stem cell self-renewal and transcriptional regulation of pluripotency. While O-GlcNAc and its requirement for embryonic development were described nearly 30 and 15 years ago, respectively, our molecular understanding of this dependence is severely lacking. Historically, this is due to the lack of strategies to directly detect O-GlcNAcylated proteins and unambiguously identify their sites of modification. The work reported here utilizes recent advancements in lectin-based O-GlcNAcylated peptide enrichment strategies, coupled with electron transfer dissociation mass spectrometry, to identify and characterize O-GlcNAc modified proteins in embryonic stem cells. This work has established that many transcriptional regulatory proteins responsible for stem cell maintenance are O-GlcNAc modified. One such example, SOX2, a transcription factor essential for establishing and maintaining pluripotency, possesses a complex modification occupancy pattern where the major form is O-GlcNAcylated. This thesis reveals that SOX2 O-GlcNAcylation is inhibitory for pluripotency transcriptional networks. Our results suggest a potential crosstalk between O-GlcNAc and phosphorylation in regulating SOX2 function, where the O-GlcNAc may prevent a nearby phosphorylation. Although O-GlcNAc signaling is essential for stem cell self- renewal we show that the connection between O-GlcNAc and transcriptional regulation, especially with respect to pluripotency, is more complex than previously appreciated.